Transpositional mutagenesis in dairy lactococci: Strategy for gene cloning
Abstract
The objective of this study was to utilize the unique characteristics of the conjugative transposon Tn1545 in the development of a strategy for insertional mutagenesis in selected dairy lactococcal strains for eventual gene targeting and cloning in an appropriate host. Transposon Tn1545 was conjugally transferred from Lactococcus lactis LM2301::Tn1545 to L. lactis GM0230 and L. lactis ssp. lactis var. diacetylactis 18-16S. Frequencies of transfer and expression of 9.3 $\times$ 10$\sp{-5}$ and 3.4 $\times$ 10$\sp{-7}$ transconjugants per donor cell, respectively, were obtained. Insertion of Tn1545 was shown to be at a specific site in L. lactis LM2301, as well as in the 28 Mdal plasmid of 18-16::Tn1545 transconjugants. A multiple or near random insertion of Tn1545 was observed in the 18-16 chromosome. Multiple insertions were also observed in some transconjugants. Cloning of Tn1545 in E. coli from an 18-16::Tn1545 transconjugant was accomplished, followed by the excision of Tn1545 from the cloned vector. The completion of the proposed strategy for transpositional mutagenesis was evidenced by the remaining lactococcal DNA junction fragments (previously flanking Tn1545) that were cloned in E. coli utilizing Tn1545 as a genetic marker for cloning. Lactococcal junction fragments were observed in the E. coli host. Cloning of Tn1545 from both plasmid and chromosome of 18-16::Tn1545 was accomplished. The cloning of lactococcal DNA in E. coli completes the strategy for insertional mutagenesis as a viable tool in the cloning of lactococcal DNA from selected strains.
Degree
Ph.D.
Advisors
Steenson, Purdue University.
Subject Area
Food science|Molecular biology
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